Abstract

The Runx family of transcription factors (Runx1, Runx2, and Runx3) are highly conserved and encode proteins involved in a variety of cell lineages, including blood and blood-related cell lineages, during developmental and adult stages of life. They perform activation and repressive functions in the regulation of gene expression. The requirement for Runx1 in the normal hematopoietic development and its dysregulation through chromosomal translocations and loss-of-function mutations as found in acute myeloid leukemias highlight the importance of this transcription factor in the healthy blood system. Whereas another review will focus on the role of Runx factors in leukemias, this review will provide an overview of the normal regulation and function of Runx factors in hematopoiesis and focus particularly on the biological effects of Runx1 in the generation of hematopoietic stem cells. We will present the current knowledge of the structure and regulatory features directing lineage-specific expression of Runx genes, the models of embryonic and adult hematopoietic development that provide information on their function, and some of the mechanisms by which they affect hematopoietic function.

Introduction

The family of Runx transcription factors (Runx1, Runx2, and Runx3) are involved in the regulation of lineage-specific genes, cell identities, and functions throughout development. Runx1 was first identified as a protein that binds in a sequence-specific manner to enhancers of Moloney murine leukemia virus1  and polyomavirus,2  and it was originally designated as core binding factor α (CBFα) and polyoma enhancer binding protein 2 (PEBP2), respectively. The gene encoding RUNX1 was cloned as one of the genes at the breakpoint of t(8,21) associated with acute myeloid leukemia, resulting in its original name AML1. Based on their homology to the Drosophila transcription factor runt, the name of this transcription factor family was changed to runt-related proteins or “Runx.”3  Each of the 3 mammalian Runx proteins contains a DNA-binding domain that is highly homologous to that of Drosophila runt. Runt domain–containing factors are found throughout the metazoan kingdom, from cnidarians and sponges to mammals, suggesting conserved roles for these factors in basic biological processes.4-6 

In mammals, Runx1 was first identified as a critical regulator of hematopoiesis,7,8  Runx2 for its role in bone development,9,10  and Runx3 for its requirement in neurogenesis.11  However, all 3 factors were later shown to play critical roles also in other organ systems,12-14  and it is now clear that not only Runx1 but also Runx2 and Runx3 play a part in hematopoiesis. Cbfβ, the heterodimeric non–DNA-binding partner protein of the Runx factors, is also critical for hematopoiesis; without it, the Runx factors cannot bind DNA efficiently to regulate expression of their target genes.15 

Within the hematopoietic system, Runx factors are expressed in a partially overlapping pattern. Runx activity as a transcription factor may be expected in all Runx-expressing cells, as its obligate partner, Cbfβ, is ubiquitously expressed.16,17  In this regard, the specific expression patterns of Runx genes predict which cell type will be affected by Runx deletion. However, where expression of the individual Runx genes overlaps, specific phenotypes may be absent due to redundancy.

The groundbreaking discovery of the requirement for Runx1 and Cbfβ in the embryonic generation of hematopoietic stem cells (HSCs) and the adult hematopoietic system7,8,15,18-21  and its role in normal adult hematopoiesis has fueled high interest and much research on this transcription factor, particularly in the regulation of clinically relevant HSCs. Here, we review the role of Runx transcription factors and Cbfβ in normal hematopoiesis, with a focus on HSCs and mammalian hematopoiesis. The involvement of Runx factors in immune cells is extensively reviewed elsewhere.22,23 

Expression and the role of Runx factors in primitive hematopoiesis

Runx1 expression is dynamic during the time when the earliest blood cells are formed, with its expression slightly preceding the appearance of blood cells (Figure 1, left). In the yolk sac, the site of origin for the primitive hematopoietic lineages (ie, primitive erythrocytes, megakaryocytes, and macrophages), Runx1 is expressed in the mesoderm of the presumptive blood islands starting from E7.5 of mouse development.19  Expression continues upon blood island mesoderm differentiation into primitive erythrocytes, but it ceases shortly after the appearance of these cells.19  It was initially reported that primitive erythrocytes do not require Runx1 for their emergence, as they are present in normal numbers in Runx1-null embryos.7,15  This was surprising, given its expression during the generation of this lineage. More recently, it was reported that primitive erythrocytes do require Runx1 for their proper maturation. In its absence, they show morphological defects, with decreased expression of the erythroid marker Ter119 and the transcription factors EKLF and Gata1.24  This is a relatively mild phenotype compared with the dramatic effect that loss of Runx1 has on later hematopoietic cells of the embryo. It is unlikely that there is Runx factor redundancy at this stage. No or very low levels of Runx3 expression were detected in these cells,25  and an impaired maturation of primitive erythrocytes was also reported in embryos carrying a Cbfβ-MYH11 knockin allele.26  Of the other lineages, primitive megakaryocyte specification still occurs in Runx1-null embryos,27  while primitive macrophages are not formed in Runx1-null embryonic stem cell (ESC) differentiation cultures28  and are severely depleted in the yolk sac.29 

Figure 1.

Embryonic sites of Runx1 expression. Diagrams of the mouse conceptus at embryonic day 7.5 (E7.5), E8.5, and E10.5 showing representative Runx1 expression patterns. Runx1 expression (blue) is measured by β-galactosidase activity in histologic sections of Runx1LacZ/+ embryos19  in which the LacZ knockin results in a Runx1 knockout allele or by immunostaining with a Runx1-specific antibody. Hematopoietic sites and cells expressing Runx1 are shown below each embryo. Runx1 expression patterns were taken from previous studies.19,28,45,60,62,64,66,67  ab, allantoic bud; bi, blood islands; BL-CFC, blast colony-forming cells; c, chorion; pl and PL, placenta; vc, vessel of confluence; ys and YS, yolk sac.

Figure 1.

Embryonic sites of Runx1 expression. Diagrams of the mouse conceptus at embryonic day 7.5 (E7.5), E8.5, and E10.5 showing representative Runx1 expression patterns. Runx1 expression (blue) is measured by β-galactosidase activity in histologic sections of Runx1LacZ/+ embryos19  in which the LacZ knockin results in a Runx1 knockout allele or by immunostaining with a Runx1-specific antibody. Hematopoietic sites and cells expressing Runx1 are shown below each embryo. Runx1 expression patterns were taken from previous studies.19,28,45,60,62,64,66,67  ab, allantoic bud; bi, blood islands; BL-CFC, blast colony-forming cells; c, chorion; pl and PL, placenta; vc, vessel of confluence; ys and YS, yolk sac.

Runx factors in yolk sac erythromyeloid and lymphoid progenitors

In contrast to the limited role for Runx1 and Cbfβ in primitive erythrocytes, both factors are critically required for the generation of all other hematopoietic cell types normally produced in the yolk sac beginning at E8 to E8.5.7,8,15,18,19,30,31  These are uni-, bi-, and multilineage hematopoietic progenitor cells (HPCs) with a generally limited lifespan,32  although specific cell types, such as erythromyeloid progenitor–derived tissue macrophages,33  B-1 B cells, and T-cell–restricted progenitors,34  can persist into adulthood. All these cell types are believed to derive from a special hemogenic subset of yolk sac endothelium in a process termed endothelial-to-hematopoietic transition (EHT).19,20,29,35,36  Yolk sac hemogenic endothelium expresses Runx119,37  (Figure 1, middle) and in the absence of Runx1 EHT is blocked, both in the embryo19,36  and in ESC differentiation cultures.38 

RUNX factors in definitive hematopoiesis

Definitive HSCs, the founders of the adult hematopoietic hierarchy, emerge first and autonomously in the major arteries (aorta, vitelline, and umbilical) of the mouse embryo. They are localized in the hematopoietic cell clusters that bud into the lumen of these vessels (Figure 1, right; and Figure 2A).39-43  The umbilical artery forms from the mesoderm in the allantois.44  Runx1 expression is seen from E7.5 in the allantois and from early E8 in endothelial cells of the nascent umbilical artery and the “vessel of confluence,” the point where the umbilical, yolk sac, and paired dorsal aortae amalgamate and from where the vitelline artery develops.45  In the mouse paired aortae, Runx1 expression starts from mid-E8 in a small subset of the endothelium, the hemogenic endothelium. Expression is initially ventral, but it later spreads to endothelial cells scattered around the entire circumference of the fused dorsal aorta and the emerging hematopoietic clusters.19,45,46  Runx1 is expressed in all emerging HPCs and HSCs in the vascular hematopoietic clusters (Figure 2A-B).19,39  Expression is also found in subaortic mesenchymal cells, starting from E10. These cells coexpress smooth muscle actin, suggesting they are developing smooth muscle cells.47,48  Finally, the early onset of Runx1 expression at all the sites of HSC emergence has implications for the interpretation of lineage-tracing experiments using tamoxifen-inducible Runx1-Cre mouse models.49,50  Together with the uncertainties about the perdurance of Cre nuclear localization, it is plausible that hemogenic endothelial cells at the sites of HSC emergence are labeled with early tamoxifen injections.

Figure 2.

Runx1 expression in the mouse dorsal aorta during EHT. (A) Diagram of the ventral wall of the mouse E10.5 aorta showing Runx1-expressing cells (blue). Cells undergoing EHT are indicated as hemogenic endothelial (he) or hematopoietic cluster (h) cells. Aortic endothelial cells are indicated in gray, and Runx1-expressing hemogenic endothelial cells are in blue. Some mesenchymal cells (beige) underlying the aorta are Runx1 expressing. Runx1-expressing hematopoietic cell clusters are closely associated with the ventral luminal wall of the aorta. (B) Runx1 in situ hybridization analysis on a transverse section through the E10.5 mouse dorsal aorta. Runx1 expression (blue) is detected mainly at the ventral aspect of the aorta where it is seen in the subaortic mesenchymal cell layer, in some endothelial cells, and in hematopoietic cluster cells. In situ hybridization, microscopy, and image processing were performed as described in Bee et al.46  Original magnification ×400. (C) Schematic of the Runx1 +23 enhancer-reporter transgene construct (23GFP) and resulting GFP expression (green) in a transverse section through the E10.5 aorta of a 23GFP transgenic embryo. Endothelial cells (VE-cadherin labeled) are in red and nuclear stain (TO-PRO-3) in blue. The 23GFP transgene marks hemogenic endothelium in the vessel wall and all hematopoietic stem and progenitor cells (HSPCs) in the aortic clusters.55,135  Immunofluorescent labeling, confocal microscopy, and image processing were performed as in Swiers et al.55  Original magnification ×300.

Figure 2.

Runx1 expression in the mouse dorsal aorta during EHT. (A) Diagram of the ventral wall of the mouse E10.5 aorta showing Runx1-expressing cells (blue). Cells undergoing EHT are indicated as hemogenic endothelial (he) or hematopoietic cluster (h) cells. Aortic endothelial cells are indicated in gray, and Runx1-expressing hemogenic endothelial cells are in blue. Some mesenchymal cells (beige) underlying the aorta are Runx1 expressing. Runx1-expressing hematopoietic cell clusters are closely associated with the ventral luminal wall of the aorta. (B) Runx1 in situ hybridization analysis on a transverse section through the E10.5 mouse dorsal aorta. Runx1 expression (blue) is detected mainly at the ventral aspect of the aorta where it is seen in the subaortic mesenchymal cell layer, in some endothelial cells, and in hematopoietic cluster cells. In situ hybridization, microscopy, and image processing were performed as described in Bee et al.46  Original magnification ×400. (C) Schematic of the Runx1 +23 enhancer-reporter transgene construct (23GFP) and resulting GFP expression (green) in a transverse section through the E10.5 aorta of a 23GFP transgenic embryo. Endothelial cells (VE-cadherin labeled) are in red and nuclear stain (TO-PRO-3) in blue. The 23GFP transgene marks hemogenic endothelium in the vessel wall and all hematopoietic stem and progenitor cells (HSPCs) in the aortic clusters.55,135  Immunofluorescent labeling, confocal microscopy, and image processing were performed as in Swiers et al.55  Original magnification ×300.

The spatiotemporal-restricted expression of Runx1 in the endothelial cells of the major embryonic arteries implicated a role for Runx1 in EHT in these locations. Indeed, in the absence of Runx1, no intra-aortic, vitelline, or umbilical artery hematopoietic cell clusters or functional HSPCs are detected.19,20,36,39  A similar requirement for Runx1 was seen in zebrafish EHT.51,52  Support for a role for Runx1 in hemogenic endothelium came from its conditional deletion in Tek- or vascular endothelial cadherin (VE-cadherin)–expressing cells. In these embryos, no hematopoietic cell clusters or functional HSCs or HPCs were formed, and embryos died around midgestation with fetal liver anemia, similar to the phenotype of Runx1-null embryos.20,35  These experiments did not distinguish between a role for Runx1 solely in the hemogenic endothelium or in the hematopoietic cluster cells, as VE-cadherin is expressed on the cluster cells and Cre could still be active there. However, Runx1 deletion in Vav-expressing hematopoietic cells did not phenocopy the Runx1 null, placing the limit of the critical Runx1 requirement prior to fetal liver colonization.20 

Tober et al demonstrated that erythromyeloid progenitors and HSCs differ in their requirement for Runx1.53  Through inducible deletion of Runx1 in VE-cadherin+ cells in 24-hour intervals, they demonstrated that progenitors require Runx1 before E10.5, while the requirement for Runx1 in the generation of HSCs extended to E11.5. Interestingly, Liakhovitskaia et al54  reported that in the absence of Runx1, there were still some CD41+ cells in the wall of the E10.5 dorsal aorta with the phenotype of pre-HSCs, suggesting that initial hematopoietic specification does still occur. Conditional rescue of Runx1 in CD41-expressing cells restored functional HSC and HPC generation. At present, it is not clear whether Runx1 rescue occurred in CD41+ pre-HSCs54  or in endothelium of the early embryo, which was previously shown to express CD41.55  The endothelium to blood fate conversion was proposed to involve downregulation of Sox17, HoxA3, and Bmi1 that act upstream of Runx1.56-58  A recent review focuses on the essential role of Runx1 in the formation of blood cells from embryonic endothelium.59 

Runx1 is also expressed in hematopoietic and endothelial cells of the placenta60,61  and in some cells of the embryonic head,62-64  2 other preliver sites where HSCs are found.60,65,66  The placenta is formed from a fusion of the chorion and allantois, both of which express Runx1 (Figure 1) and exhibit hematopoietic potential ex vivo.67  While the placenta generates multilineage progenitors de novo,61  it remains to be established whether HSCs are also generated in this tissue. In the human placenta, HSCs appear later than in the AGM, suggesting that this tissue is seeded by HSCs.68,69  HSC and HPC generation in head vasculature has proven elusive, with no evidence of EHT being reported.62-64 

Interestingly, Runx1 haploinsufficiency during mouse development results in a dramatic change in the spatiotemporal distribution of HSCs, with a premature appearance of HSCs in the aorta and yolk sac.18  In mouse ESC differentiation cultures, Runx1 haploinsufficiency accelerates mesodermal development and hemangioblast specification.70  A gene regulatory network of a transcription factor triad (Scl, Gata2, and Fli1) that responds to Notch1 and BMP4 (through Runx1) signaling has been implicated by mathematical modeling to be involved in the accelerated emergence of HSCs when only 1 allele of Runx1 is expressed. In a simulation, the decrease in activation threshold in the haploinsufficient state results in an accelerated (∼2-day) triad activation.71 

Only Runx1 plays a critical role at the onset of definitive hematopoiesis. Runx2 or Runx3 expression has not been reported in the aorta, vitelline, and umbilical arteries during EHT, and deletion of Runx29  or Runx372,73  did not result in the midgestation embryonic lethality and fetal liver anemia seen in Runx1-null embryos. In contrast, germline deletion of Cbfβ results in a phenotype similar to that of Runx1 germline deletion. Embryos die at E12.5 with fetal liver anemia and lack HSCs.15,30,31  Remarkably, Cbfβ-null fetal livers still contain a small number of HPCs, suggestive of a Cfbβ-independent role for Runx1 in early hematopoiesis, similar to that reported in zebrafish.74 

RUNX factors in fetal and adult hematopoiesis

All blood cells in the adult (HSCs, HPCs, and mature blood cells) except erythrocytes express Runx1.75,76  MX-Cre conditional deletion of Runx1 in bone marrow (BM) chimeras showed an increase in lineage marker–negative, Sca1-positive, Kit-positive (LSK) cells, granulocytes, myeloid progenitors, and megakaryocyte progenitors, and T lymphocyte development was blocked.77-80  It was thought that Runx1 plays a role in regulating homeostatic HSC and HPC numbers in the BM. Although discrepancies in the phenotypic marker expression on hematopoietic cells were found in the absence of Runx1,77  long-term reconstituting (LTR) HSC frequency was three- to fourfold decreased as determined by peripheral blood chimerism. However, there was no change in BM chimerism, and thus, it was concluded that there is minimal impact of Runx1 on adult BM LTR HSCs.

Vav-Cre conditional deletion was used to examine whether HSCs are dependent on Runx1 and Cbfβ after they are generated. The onset of Vav-Cre activity is at E11.5 in HSCs.20  Whereas the deletion of Runx1 showed no significant reductions in phenotypic HSC or LTR HSCs in the E14.5 fetal liver,77 CBFβ deletion severely compromises LTR HSCs.53  Thus, Runx2 and/or Runx3 are likely able to contribute to the maintenance of HSCs. These results, together with the fact that Runx3 is expressed in fetal liver hematopoietic precursors and partially overlaps with Runx1 expression,81  support the notion that Runx family proteins act in a redundant manner in HSCs after they are generated.

Interestingly, the double deletion of Runx1 and Runx3 (MX-Cre) in adult mice was found to lead to BM failure (Fanconi anemia–like) and myeloproliferative disease.82  BM failure was found to be the result of differentiation blocks in all hematopoietic lineages and progressive HSC exhaustion. DNA repair was found to be defective, and it was concluded that repair relies on Runx-dependent recruitment of Fanconi proteins to the site of DNA damage. This activity is independent of Cbfβ, suggesting that Runx1 and Runx3 may function here in a non–transcription-related manner.

Haploinsufficiency of Runx1 also affects HSCs in the adult BM. LTR-HSCs are decreased by 50%, and upon transplantation, these HSCs provided enhanced engraftment and normal numbers of most hematopoietic lineages as compared with wild-type mice. In contrast, a decrease in CD4+ T cells and circulating platelets was observed.83  Runx1 haploinsufficient myeloid progenitors were found to be differentiation impaired. Although granulocyte lineage commitment occurs, progenitors do not mature84  and are hypersensitive to granulocyte colony-stimulating factor, predisposing the progenitors to expansion and mobilization rather than differentiation.85  Similarly, in humans, autosomal-dominant mutations in 1 Runx1 allele result in qualitative and quantitative platelet defects and a propensity to develop acute myeloid leukemia.86  Most recently, Vav-Cre–mediated deletion of Runx1 has been found to result in an increase in the stress resistance of BM HSCs and the superior survival of HSCs in the presence of genotoxic shock. Runx1 binds to promoters of genes encoding ribosome proteins, and in its absence, ribosome biogenesis is reduced,87  suggesting that this condition may facilitate the premalignant cell state.

Runx1 and Cbfβ have been found to also play a role in the regulation of pro–B cells and double-negative thymocytes, and the compound disruption of Runx1 and Runx3 genes suggests partially redundant function.88-90  Runx1 is expressed in earliest developing thymocytes and later in cortical thymocytes at E18 and postnatal week 6.90  Its expression is highest in the most immature thymocytes,91  supporting a role in T-cell lineage fate decisions. In the adult, Foxp3+ regulatory T cells and Th17 cells73  express Runx1. It is also expressed in Flt3+ dendritic cell precursors and common lymphoid progenitors, and Cbfβ is critical for the differentiation of these cells.92 

Runx3 plays a role in innate immune cell types and T lymphoid development. Runx3-deficient mice exhibit defects in splenic natural killer cells, lack Langerhans and dendritic epithelial cells,72,73  and the lineage commitment of innate lymphoid cells is affected.93  At E18, Runx3 is expressed in thymocytes (medulla and cortical) and in the adult it is detected mainly in the medulla.90 Runx3 null mice exhibit defects in the differentiation of CD8+ cytotoxic T cells and Th1 cells.72,73  It is most highly expressed in mature thymocytes91  and is thought to act together with Runx1 as a transcriptional repressor of CD4 during T-cell lineage decisions.

Although Runx2 expression is high in the HSPC compartment of mouse BM,94  there is no data thus far demonstrating a role for Runx2 in fetal or adult BM HSCs.

Runx1 mechanism of action

Given the critical role of Runx1 in the establishment and maintenance of the adult hematopoietic system, there has been much interest in elucidating how it exerts it role. Runx1 acts as an epigenetic regulator and as an activator or repressor of transcriptional programs (reviewed in Tober et al,59 Lausen,95  Lichtinger et al,96  Obier and Bonifer,97  and Swiers et al98). In the dorsal aorta, Runx1 drives hematopoietic specification, which coincides with loss of endothelial potential.55-58  In ESC hematopoietic differentiation cultures, Runx1 is involved in the initial remodeling of the Pu.1 transcription factor locus, and Pu.1 is one of its important downstream targets.99,100  Intriguingly, this required only low-level transient expression of Runx1. Genome-wide analysis showed that in hemogenic endothelium, binding of SCL, Fli1, and Cebpβ was associated with the priming of a large number of hematopoietic loci. Upon upregulation of Runx1, these complexes relocated to new cis elements, now bound by Runx1 and showing increased histone acetylation. Thus, Runx1 changes the epigenetic landscape at hematopoietic loci to promote hematopoietic differentiation.101 

Runx1 expression in ESC-derived hemogenic endothelium induces adhesion and migration-associated transcriptional profiles before the downregulation of endothelial genes.102  The Runx1 targets Gfi1 and Gfi1b are implicated in the downregulation of the endothelial program.103  They do this through recruitment of the chromatin modifier LSD1 (a member of the CoREST repressive complex) to endothelial gene loci.104  Later in hematopoietic differentiation, Runx1 positively regulates the expression of a large number of hematopoietic lineage–specific genes, including cytokines, cytokine receptors, and T-cell receptors,23,59,98  many of which are effectors of hematopoietic fate, differentiation, and function. Whether Runx1 functions as a transcriptional activator or repressor is dependent upon cofactor interactions. Posttranslational modifications by tyrosyl phosphorylation regulate the interactions of Runx1 with different coregulators.105 

Structure of mammalian Runx genes and alternatively spliced isoforms

The genomic organization of mouse and human Runx genes is highly conserved. All vertebrate Runx genes have 2 promoters (distal P1 and proximal P2) and a variable number of alternatively spliced protein-coding exons (Figure 3).4,5,106-110  P1- and P2-derived alternative transcripts are differentially expressed in various cell types and developmental stages. Below, we focus on Runx1 as the main regulator of hematopoiesis, but also Runx2 and Runx3 express transcripts from 2 promoters to generate alternative isoforms.94,111-113 

Figure 3.

Runx1 locus and isoforms. (A) Schematic of the 224-kb mouse Runx1 locus, alternative splice forms, and the +23 enhancer (not to scale) reported in the literature.46,108,109,114,128,142,143  White boxes, coding sequence; red boxes, untranslated regions (UTRs); blue box, predicted coding +UTR. Figure 3A reprinted with minimal alteration from Bee et al46  with permission from Elsevier. (B) Schematic showing Runx1 protein domains relative to their coding exons.110  CDS, coding sequence; ID, inhibitory domain; RD, runt domain; TAD, transactivation domain.

Figure 3.

Runx1 locus and isoforms. (A) Schematic of the 224-kb mouse Runx1 locus, alternative splice forms, and the +23 enhancer (not to scale) reported in the literature.46,108,109,114,128,142,143  White boxes, coding sequence; red boxes, untranslated regions (UTRs); blue box, predicted coding +UTR. Figure 3A reprinted with minimal alteration from Bee et al46  with permission from Elsevier. (B) Schematic showing Runx1 protein domains relative to their coding exons.110  CDS, coding sequence; ID, inhibitory domain; RD, runt domain; TAD, transactivation domain.

Alternative promotor usage contributes to regulatory complexity, with the production of Runx1 transcripts that differ in their 5′ and 3′ UTRs, their coding sequence, and the time required for their transcription (Figure 3).46,81,106,114  Differences in the identity and length of the UTRs have been linked to differences in RNA stability and translation efficiency.115,116  P1-derived transcripts are subject to Cap-mediated translation, while P2-derived transcripts undergo internal ribosome entry site–mediated translation. The latter is thought to facilitate translation under conditions of stress and during cell division.116  Translation of Runx1 is also subject to regulation by microRNAs.117,118  Several alternatively spliced transcripts have been described in mouse (Figure 3) and human,119  including the full-length P1- and P2-derived Runx1c and Runx1b. One of these transcripts, RUNX1a, lacks the transactivation domain while retaining the DNA-binding domain and has been proposed to act as a dominant negative.119,120 

The biological relevance of the different Runx1 isoforms is not fully understood. Exclusive expression of the full-length Runx1 is sufficient for in vitro hematopoiesis and for embryos to develop into adulthood,28,75,121,122  and both the runt domain and the transactivation domain are essential.121,122  As no detailed functional analyses of HSCs and HPCs in vivo were performed, the precise role of this and other Runx1 isoforms in HSC biology remained unclear. More recently, enforced expression of the short human RUNX1a isoform was shown to favor expansion of the HSC pool, in line with earlier reports that it supports proliferation in a myeloid cell line.123-126  Expression of the full-length RUNX1b and RUNX1c, in contrast, promoted differentiation, depleting the HSC pool. A similar short isoform lacking the transactivation domain was described in mice, albeit with a different 3′ exon (Figure 3).127,128  Interestingly, the mouse short Runx1 isoform had a biological role similar to that of the human RUNX1a in that it positively regulated the size of the HSC pool.127 

The full-length P1- and P2-derived Runx1c and Runx1b protein isoforms are identical apart from their N terminus. In human and mouse, there are 32 and 19 unique RUNX1c N-terminal amino acids, respectively, and 5 unique RUNX1b ones. In the mouse, no differences in hematopoietic development were observed in ESC differentiation cultures upon overexpression of Runx1c or Runx1b.129  In human, however, a recent study reported on a specific role for the RUNX1c in regulating the growth of Epstein-Barr virus–transformed B cells, unearthing a first indication for specific functionality associated with this domain. Interestingly, the sequence responsible for this effect is specific to the human RUNX1c N terminus.130 

Tissue-specific expression of RUNX

All 3 Runx proteins bind the same 5′-ACCG/ACA-3′ DNA motif and their specific roles are due to their spatiotemporal expression patterns. Both promoters of human and mouse RUNX1 contain consensus binding motifs for several hematopoietic transcription factors (E-box, Ets, and Runx), but these are not sufficient to mediate tissue-specific transgene expression.106,131  The P2 promoter lies in a large CpG island, often found at promoters of broadly expressed genes. The P1 promoter, which shows greater tissue-specific activity than P2,107  contains 2 adjacent conserved Runx motifs just downstream of the transcription start site (conserved in all 3 Runx genes). These play a role in autoregulation of Runx1 during hematopoietic development and in auto- and crossregulation in the adult (reviewed in Bee et al46 ). Interestingly, Smad1, a positive mediator of Bmp4 signaling, was found to transactivate the Runx1 P1 promoter, whereas Smad6 acts as an inhibitor.132  Also, the Wnt pathway was reported to activate the RUNX1 P1 promoter.133 

Runx1 promoters show specific activity patterns during developmental differentiation of cells to the hematopoietic lineage. At all sites of HSPC emergence, P2 promoter activity precedes that of P1.46,129,134,135  The P1 promoter is activated when hemogenic cells commit to a hematopoietic fate. As hematopoietic development proceeds in the fetal liver, P1 becomes the dominant promoter and remains so in adult BM.46  A similar switch in promoter usage is seen in the developing thymus.136  Mouse and human ESCs undergoing hematopoietic differentiation also show P2 promoter usage before P1.126,134,137,138  The sequential P2 to P1 promoter usage may reflect a more ancient role for P2 (present in all metazoan Runx genes) than P1, which is vertebrate specific.4,5 

Although the Runx1 promoters do not contribute tissue specificity, they play important, nonredundant roles in hematopoiesis. Analysis of a P1-null mouse model showed that lack of Runx1c negatively affects the initial emergence of HPCs in the embryo, but embryos survive into adulthood.135  Adult P1-null mice show a phenotype in between Runx1 haploinsufficient and Runx1 MX-CRE–deleted mice.135  Loss of P1 also leads to severely reduced numbers of basophils and T-cell lineage defects.139,140  In an attenuated P2 promoter mouse model, embryos die around midgestation with hemorrhages, fetal liver anemia, and defects in thymocyte development.136  The generation of hematopoietic progenitors and aortic cell clusters is severely affected,135  and this phenotype is in line with the earlier and broader expression of P2-derived isoforms. Thus, both Runx1 promoters contribute to the expression of normal spatiotemporal levels of Runx1. How alternative promoter expression is regulated is poorly understood. A study in zebrafish suggests a role for cohesion and CTCF.141 

The finding that Runx1 promoters do not direct tissue-specific expression prompted the search for enhancers that mediate Runx1 expression during EHT and in HSCs. Using a combination of comparative genomics, chromatin accessibility assays, and transient mouse transgenesis, a highly conserved hematopoietic Runx1 +23 enhancer was identified in the first intron of the 224-kb mouse gene.142  It was shown that the +23 enhancer is sufficient to mediate reporter gene expression in a pattern similar to Runx1 during hematopoietic development. It directs reporter gene expression in all emerging HSCs and HPCs and labels all hematopoietic-fated endothelial cells in the mouse embryo (Figure 2C).55,135,142,143  Of note, it does not mark the subaortic Runx1+ smooth muscle cells or any other nonhematopoietic Runx1-expressing tissues in the embryo.55,142  The mouse +23 enhancer was reported to also mark hematopoietic-fated endothelial cells and hematopoietic cells in zebrafish,143-145  although it is not conserved in this species.131  In human ESC and induced pluripotent stem cell differentiation cultures, the +23 enhancer in conjunction with the RUNX1 P1 promoter is active in hematopoietic cells and in colony-forming units in culture.146  Thus, the Runx1 +23 enhancer is a valuable tool in studies examining hematopoietic development.

Insight into the pathways converging on Runx1 will yield important information on signals underlying hematopoietic specification. A recent review59  summarizes the known transcription factors and pathways acting upstream of Runx1. Some of these act directly on the Runx1 +23 enhancer. Indeed, +23 enhancer activity is dependent on Gata, Runx, and Ets motifs; Gata2, Ets factors (Fli-1, Elf-1, and Pu.1), and the SCL/Lmo2/Ldb1 complex are bound to the enhancer in vivo in yolk sac and fetal liver.25,142  In addition to the +23 enhancer, several other hematopoietic Runx1 enhancers also mediate reporter gene expression to the dorsal aorta and fetal liver in a Runx-specific pattern.147  Most of these also contain binding motifs for Gata, Runx, and Ets and are bound by these and other hematopoietic transcription factors. The precise regulatory roles of these enhancers on Runx1 expression in developmental hematopoiesis, and the developmental pathways converging on them, remain to be established.

Implications and perspectives

Several decades of research on the Runx protein family has provided a wealth of molecular and biological information concerning their roles in normal hematopoiesis. Their many isoforms and expression patterns highlight a complexity that goes beyond a simple description of 3 transcription factors. The overlapping expression of Runx1 and Runx3 in some hematopoietic cell lineages reveals functional redundancy. As all 3 Runx proteins interact with their obligate partner, Cbfβ, deletion of Cbfβ has more profound consequences for hematopoietic cells than single Runx gene deletions. Novel mouse models reveal the mechanisms underlying Runx function, such as in ribosome biosynthesis, which in Runx1 deficiency affects the stress status of HSCs. These long-term in vivo studies are for the first time providing an understanding of the preleukemic condition that affects the balance of Runx-centric gene regulatory networks and epigenetic status. Moreover, roles for Runx proteins outside of their classification as transcription factors are being proposed, such as in DNA repair in which Runx acts independently of Cbfβ.

As the Runx field moves forward, new methods of single-cell analysis are being employed. Time-lapse images of single Runx1-expressing hematopoietic cells during emergence from hemogenic endothelium confirmed EHT and in the future are likely to provide important information on the dynamic expression of Runx1, transcription factor assemblies, and reshaping of the epigenetic landscape during this process. Transcriptomic and proteomic signatures are expected to elucidate the inherent instability of hematopoietic cells and their heterogeneity during hematopoietic cell fate transitions in development and adult hematopoietic differentiation and during aging. Together, this and future knowledge on Runx proteins will benefit biomedical approaches for the treatment of Runx-related hematologic dysfunctions and pathologies.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank Chris S. Vink and Claudia Baumann for the in situ hybridization and immunostained images in Figure 2.

M.d.B. is supported by an MRC Molecular Hematology Unit (Oxford) Core Award. E.D. is supported by ZonMW TOP (91211068) and a European Research Council Advanced Grant (341096).

Authorship

Contribution: M.d.B. and E.D. reviewed the literature and wrote the review.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Elaine Dzierzak, University of Edinburgh, Centre for Inflammation Research, Queens Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom; e-mail: elaine.dzierzak@ed.ac.uk.

References

References
1.
Speck
NA
,
Baltimore
D
.
Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemia virus enhancer
.
Mol Cell Biol
.
1987
;
7
(
3
):
1101
-
1110
.
2.
Kamachi
Y
,
Ogawa
E
,
Asano
M
, et al
.
Purification of a mouse nuclear factor that binds to both the A and B cores of the polyomavirus enhancer
.
J Virol
.
1990
;
64
(
10
):
4808
-
4819
.
3.
van Wijnen
AJ
,
Stein
GS
,
Gergen
JP
, et al
.
Nomenclature for Runt-related (RUNX) proteins
.
Oncogene
.
2004
;
23
(
24
):
4209
-
4210
.
4.
Levanon
D
,
Groner
Y
.
Structure and regulated expression of mammalian RUNX genes
.
Oncogene
.
2004
;
23
(
24
):
4211
-
4219
.
5.
Rennert
J
,
Coffman
JA
,
Mushegian
AR
,
Robertson
AJ
.
The evolution of Runx genes I. A comparative study of sequences from phylogenetically diverse model organisms
.
BMC Evol Biol
.
2003
;
3
:
4
.
6.
Sullivan
JC
,
Sher
D
,
Eisenstein
M
, et al
.
The evolutionary origin of the Runx/CBFbeta transcription factors--studies of the most basal metazoans
.
BMC Evol Biol
.
2008
;
8
:
228
.
7.
Okuda
T
,
van Deursen
J
,
Hiebert
SW
,
Grosveld
G
,
Downing
JR
.
AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis
.
Cell
.
1996
;
84
(
2
):
321
-
330
.
8.
Wang
Q
,
Stacy
T
,
Binder
M
,
Marin-Padilla
M
,
Sharpe
AH
,
Speck
NA
.
Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis
.
Proc Natl Acad Sci USA
.
1996
;
93
(
8
):
3444
-
3449
.
9.
Komori
T
,
Yagi
H
,
Nomura
S
, et al
.
Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts
.
Cell
.
1997
;
89
(
5
):
755
-
764
.
10.
Otto
F
,
Thornell
AP
,
Crompton
T
, et al
.
Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development
.
Cell
.
1997
;
89
(
5
):
765
-
771
.
11.
Levanon
D
,
Bettoun
D
,
Harris-Cerruti
C
, et al
.
The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons
.
EMBO J
.
2002
;
21
(
13
):
3454
-
3463
.
12.
Blyth
K
,
Vaillant
F
,
Jenkins
A
, et al
.
Runx2 in normal tissues and cancer cells: A developing story
.
Blood Cells Mol Dis
.
2010
;
45
(
2
):
117
-
123
.
13.
Liakhovitskaia
A
,
Lana-Elola
E
,
Stamateris
E
,
Rice
DP
,
van ’t Hof
RJ
,
Medvinsky
A
.
The essential requirement for Runx1 in the development of the sternum
.
Dev Biol
.
2010
;
340
(
2
):
539
-
546
.
14.
Lotem
J
,
Levanon
D
,
Negreanu
V
, et al
.
Runx3 at the interface of immunity, inflammation and cancer
.
Biochim Biophys Acta
.
2015
;
1855
(
2
):
131
-
143
.
15.
Wang
Q
,
Stacy
T
,
Miller
JD
, et al
.
The CBFbeta subunit is essential for CBFalpha2 (AML1) function in vivo
.
Cell
.
1996
;
87
(
4
):
697
-
708
.
16.
Ogawa
E
,
Inuzuka
M
,
Maruyama
M
, et al
.
Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2 alpha
.
Virology
.
1993
;
194
(
1
):
314
-
331
.
17.
Wang
S
,
Wang
Q
,
Crute
BE
,
Melnikova
IN
,
Keller
SR
,
Speck
NA
.
Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor
.
Mol Cell Biol
.
1993
;
13
(
6
):
3324
-
3339
.
18.
Cai
Z
,
de Bruijn
M
,
Ma
X
, et al
.
Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo
.
Immunity
.
2000
;
13
(
4
):
423
-
431
.
19.
North
T
,
Gu
TL
,
Stacy
T
, et al
.
Cbfa2 is required for the formation of intra-aortic hematopoietic clusters
.
Development
.
1999
;
126
(
11
):
2563
-
2575
.
20.
Chen
MJ
,
Yokomizo
T
,
Zeigler
BM
,
Dzierzak
E
,
Speck
NA
.
Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter
.
Nature
.
2009
;
457
(
7231
):
887
-
891
.
21.
Zovein
AC
,
Hofmann
JJ
,
Lynch
M
, et al
.
Fate tracing reveals the endothelial origin of hematopoietic stem cells
.
Cell Stem Cell
.
2008
;
3
(
6
):
625
-
636
.
22.
de Bruijn
MF
,
Speck
NA
.
Core-binding factors in hematopoiesis and immune function
.
Oncogene
.
2004
;
23
(
24
):
4238
-
4248
.
23.
Voon
DC
,
Hor
YT
,
Ito
Y
.
The RUNX complex: reaching beyond haematopoiesis into immunity
.
Immunology
.
2015
;
146
(
4
):
523
-
536
.
24.
Yokomizo
T
,
Hasegawa
K
,
Ishitobi
H
, et al
.
Runx1 is involved in primitive erythropoiesis in the mouse
.
Blood
.
2008
;
111
(
8
):
4075
-
4080
.
25.
Landry
JR
,
Kinston
S
,
Knezevic
K
, et al
.
Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver
.
Blood
.
2008
;
111
(
6
):
3005
-
3014
.
26.
Castilla
LH
,
Wijmenga
C
,
Wang
Q
, et al
.
Failure of embryonic hematopoiesis and lethal hemorrhages in mouse embryos heterozygous for a knocked-in leukemia gene CBFB-MYH11
.
Cell
.
1996
;
87
(
4
):
687
-
696
.
27.
Potts
KS
,
Sargeant
TJ
,
Markham
JF
, et al
.
A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo
.
Blood
.
2014
;
124
(
17
):
2725
-
2729
.
28.
Lacaud
G
,
Gore
L
,
Kennedy
M
, et al
.
Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro
.
Blood
.
2002
;
100
(
2
):
458
-
466
.
29.
Frame
JM
,
Fegan
KH
,
Conway
SJ
,
McGrath
KE
,
Palis
J
.
Definitive hematopoiesis in the yolk sac emerges from Wnt-responsive hemogenic endothelium independently of circulation and arterial identity
.
Stem Cells
.
2016
;
34
(
2
):
431
-
444
.
30.
Niki
M
,
Okada
H
,
Takano
H
, et al
.
Hematopoiesis in the fetal liver is impaired by targeted mutagenesis of a gene encoding a non-DNA binding subunit of the transcription factor, polyomavirus enhancer binding protein 2/core binding factor
.
Proc Natl Acad Sci USA
.
1997
;
94
(
11
):
5697
-
5702
.
31.
Sasaki
K
,
Yagi
H
,
Bronson
RT
, et al
.
Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta
.
Proc Natl Acad Sci USA
.
1996
;
93
(
22
):
12359
-
12363
.
32.
Palis
J
.
Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo
.
FEBS Lett
.
2016
;
590
(
22
):
3965
-
3974
.
33.
Perdiguero
EG
,
Klapproth
K
,
Schulz
C
, et al
.
The origin of tissue-resident macrophages: when an erythro-myeloid progenitor is an erythro-myeloid progenitor
.
Immunity
.
2015
;
43
(
6
):
1023
-
1024
.
34.
Lin
Y
,
Yoder
MC
,
Yoshimoto
M
.
Lymphoid progenitor emergence in the murine embryo and yolk sac precedes stem cell detection
.
Stem Cells Dev
.
2014
;
23
(
11
):
1168
-
1177
.
35.
Li
Z
,
Chen
MJ
,
Stacy
T
,
Speck
NA
.
Runx1 function in hematopoiesis is required in cells that express Tek
.
Blood
.
2006
;
107
(
1
):
106
-
110
.
36.
Yokomizo
T
,
Ogawa
M
,
Osato
M
, et al
.
Requirement of Runx1/AML1/PEBP2alphaB for the generation of haematopoietic cells from endothelial cells
.
Genes Cells
.
2001
;
6
(
1
):
13
-
23
.
37.
Padrón-Barthe
L
,
Temiño
S
,
Villa del Campo
C
,
Carramolino
L
,
Isern
J
,
Torres
M
.
Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo
.
Blood
.
2014
;
124
(
16
):
2523
-
2532
.
38.
Lancrin
C
,
Sroczynska
P
,
Serrano
AG
, et al
.
Blood cell generation from the hemangioblast
.
J Mol Med (Berl)
.
2010
;
88
(
2
):
167
-
172
.
39.
North
TE
,
de Bruijn
MF
,
Stacy
T
, et al
.
Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo
.
Immunity
.
2002
;
16
(
5
):
661
-
672
.
40.
de Bruijn
MF
,
Ma
X
,
Robin
C
,
Ottersbach
K
,
Sanchez
MJ
,
Dzierzak
E
.
Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta
.
Immunity
.
2002
;
16
(
5
):
673
-
683
.
41.
de Bruijn
MF
,
Speck
NA
,
Peeters
MC
,
Dzierzak
E
.
Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo
.
EMBO J
.
2000
;
19
(
11
):
2465
-
2474
.
42.
Medvinsky
A
,
Dzierzak
E
.
Definitive hematopoiesis is autonomously initiated by the AGM region
.
Cell
.
1996
;
86
(
6
):
897
-
906
.
43.
Müller
AM
,
Medvinsky
A
,
Strouboulis
J
,
Grosveld
F
,
Dzierzak
E
.
Development of hematopoietic stem cell activity in the mouse embryo
.
Immunity
.
1994
;
1
(
4
):
291
-
301
.
44.
Downs
KM
,
Gifford
S
,
Blahnik
M
,
Gardner
RL
.
Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis
.
Development
.
1998
;
125
(
22
):
4507
-
4520
.
45.
Daane
JM
,
Downs
KM
.
Hedgehog signaling in the posterior region of the mouse gastrula suggests manifold roles in the fetal-umbilical connection and posterior morphogenesis
.
Dev Dyn
.
2011
;
240
(
9
):
2175
-
2193
.
46.
Bee
T
,
Liddiard
K
,
Swiers
G
, et al
.
Alternative Runx1 promoter usage in mouse developmental hematopoiesis
.
Blood Cells Mol Dis
.
2009
;
43
(
1
):
35
-
42
.
47.
Mirshekar-Syahkal
B
,
Haak
E
,
Kimber
GM
, et al
.
Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells
.
Haematologica
.
2013
;
98
(
2
):
163
-
171
.
48.
Swiers
G
,
Rode
C
,
Azzoni
E
,
de Bruijn
MF
.
A short history of hemogenic endothelium
.
Blood Cells Mol Dis
.
2013
;
51
(
4
):
206
-
212
.
49.
Samokhvalov
IM
,
Samokhvalova
NI
,
Nishikawa
S
.
Cell tracing shows the contribution of the yolk sac to adult haematopoiesis
.
Nature
.
2007
;
446
(
7139
):
1056
-
1061
.
50.
Tanaka
Y
,
Hayashi
M
,
Kubota
Y
, et al
.
Early ontogenic origin of the hematopoietic stem cell lineage
.
Proc Natl Acad Sci USA
.
2012
;
109
(
12
):
4515
-
4520
.
51.
Kissa
K
,
Herbomel
P
.
Blood stem cells emerge from aortic endothelium by a novel type of cell transition
.
Nature
.
2010
;
464
(
7285
):
112
-
115
.
52.
Lam
EY
,
Hall
CJ
,
Crosier
PS
,
Crosier
KE
,
Flores
MV
.
Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells
.
Blood
.
2010
;
116
(
6
):
909
-
914
.
53.
Tober
J
,
Yzaguirre
AD
,
Piwarzyk
E
,
Speck
NA
.
Distinct temporal requirements for Runx1 in hematopoietic progenitors and stem cells
.
Development
.
2013
;
140
(
18
):
3765
-
3776
.
54.
Liakhovitskaia
A
,
Rybtsov
S
,
Smith
T
, et al
.
Runx1 is required for progression of CD41+ embryonic precursors into HSCs but not prior to this
.
Development
.
2014
;
141
(
17
):
3319
-
3323
.
55.
Swiers
G
,
Baumann
C
,
O’Rourke
J
, et al
.
Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level
.
Nat Commun
.
2013
;
4
:
2924
.
56.
Eliades
A
,
Wareing
S
,
Marinopoulou
E
, et al
.
The hemogenic competence of endothelial progenitors is restricted by Runx1 silencing during embryonic development
.
Cell Reports
.
2016
;
15
(
10
):
2185
-
2199
.
57.
Iacovino
M
,
Chong
D
,
Szatmari
I
, et al
.
HoxA3 is an apical regulator of haemogenic endothelium
.
Nat Cell Biol
.
2011
;
13
(
1
):
72
-
78
.
58.
Lizama
CO
,
Hawkins
JS
,
Schmitt
CE
, et al
.
Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition
.
Nat Commun
.
2015
;
6
:
7739
.
59.
Tober
J
,
Maijenburg
MW
,
Speck
NA
.
Taking the leap: Runx1 in the formation of blood from endothelium
.
Curr Top Dev Biol
.
2016
;
118
:
113
-
162
.
60.
Ottersbach
K
,
Dzierzak
E
.
The murine placenta contains hematopoietic stem cells within the vascular labyrinth region
.
Dev Cell
.
2005
;
8
(
3
):
377
-
387
.
61.
Rhodes
KE
,
Gekas
C
,
Wang
Y
, et al
.
The emergence of hematopoietic stem cells is initiated in the placental vasculature in the absence of circulation
.
Cell Stem Cell
.
2008
;
2
(
3
):
252
-
263
.
62.
Iizuka
K
,
Yokomizo
T
,
Watanabe
N
, et al
.
Lack of phenotypical and morphological evidences of endothelial to hematopoietic transition in the murine embryonic head during hematopoietic stem cell emergence
.
PLoS One
.
2016
;
11
(
5
):
e0156427
.
63.
Li
Z
,
Vink
CS
,
Mariani
SA
,
Dzierzak
E
.
Subregional localization and characterization of Ly6aGFP-expressing hematopoietic cells in the mouse embryonic head
.
Dev Biol
.
2016
;
416
(
1
):
34
-
41
.
64.
Yzaguirre
AD
,
Speck
NA
.
Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites
.
Dev Dyn
.
2016
;
245
(
10
):
1011
-
1028
.
65.
Gekas
C
,
Dieterlen-Lièvre
F
,
Orkin
SH
,
Mikkola
HK
.
The placenta is a niche for hematopoietic stem cells
.
Dev Cell
.
2005
;
8
(
3
):
365
-
375
.
66.
Li
Z
,
Lan
Y
,
He
W
, et al
.
Mouse embryonic head as a site for hematopoietic stem cell development
.
Cell Stem Cell
.
2012
;
11
(
5
):
663
-
675
.
67.
Zeigler
BM
,
Sugiyama
D
,
Chen
M
,
Guo
Y
,
Downs
KM
,
Speck
NA
.
The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential
.
Development
.
2006
;
133
(
21
):
4183
-
4192
.
68.
Ivanovs
A
,
Rybtsov
S
,
Welch
L
,
Anderson
RA
,
Turner
ML
,
Medvinsky
A
.
Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region
.
J Exp Med
.
2011
;
208
(
12
):
2417
-
2427
.
69.
Robin
C
,
Bollerot
K
,
Mendes
S
, et al
.
Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development
.
Cell Stem Cell
.
2009
;
5
(
4
):
385
-
395
.
70.
Lacaud
G
,
Kouskoff
V
,
Trumble
A
,
Schwantz
S
,
Keller
G
.
Haploinsufficiency of Runx1 results in the acceleration of mesodermal development and hemangioblast specification upon in vitro differentiation of ES cells
.
Blood
.
2004
;
103
(
3
):
886
-
889
.
71.
Narula
J
,
Williams
CJ
,
Tiwari
A
,
Marks-Bluth
J
,
Pimanda
JE
,
Igoshin
OA
.
Mathematical model of a gene regulatory network reconciles effects of genetic perturbations on hematopoietic stem cell emergence
.
Dev Biol
.
2013
;
379
(
2
):
258
-
269
.
72.
Levanon
D
,
Negreanu
V
,
Lotem
J
, et al
.
Transcription factor Runx3 regulates interleukin-15-dependent natural killer cell activation
.
Mol Cell Biol
.
2014
;
34
(
6
):
1158
-
1169
.
73.
Wong
WF
,
Kohu
K
,
Chiba
T
,
Sato
T
,
Satake
M
.
Interplay of transcription factors in T-cell differentiation and function: the role of Runx
.
Immunology
.
2011
;
132
(
2
):
157
-
164
.
74.
Bresciani
E
,
Carrington
B
,
Wincovitch
S
, et al
.
CBFβ and RUNX1 are required at 2 different steps during the development of hematopoietic stem cells in zebrafish
.
Blood
.
2014
;
124
(
1
):
70
-
78
.
75.
Lorsbach
RB
,
Moore
J
,
Ang
SO
,
Sun
W
,
Lenny
N
,
Downing
JR
.
Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression
.
Blood
.
2004
;
103
(
7
):
2522
-
2529
.
76.
North
TE
,
Stacy
T
,
Matheny
CJ
,
Speck
NA
,
de Bruijn
MF
.
Runx1 is expressed in adult mouse hematopoietic stem cells and differentiating myeloid and lymphoid cells, but not in maturing erythroid cells
.
Stem Cells
.
2004
;
22
(
2
):
158
-
168
.
77.
Cai
X
,
Gaudet
JJ
,
Mangan
JK
, et al
.
Runx1 loss minimally impacts long-term hematopoietic stem cells
.
PLoS One
.
2011
;
6
(
12
):
e28430
.
78.
Growney
JD
,
Shigematsu
H
,
Li
Z
, et al
.
Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype
.
Blood
.
2005
;
106
(
2
):
494
-
504
.
79.
Ichikawa
M
,
Asai
T
,
Saito
T
, et al
.
AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis
.
Nat Med
.
2004
;
10
(
3
):
299
-
304
.
80.
Behrens
K
,
Triviai
I
,
Schwieger
M
, et al
.
Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions
.
Blood
.
2016
;
127
(
26
):
3369
-
3381
.
81.
Levanon
D
,
Brenner
O
,
Negreanu
V
, et al
.
Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis
.
Mech Dev
.
2001
;
109
(
2
):
413
-
417
.
82.
Wang
CQ
,
Krishnan
V
,
Tay
LS
, et al
.
Disruption of Runx1 and Runx3 leads to bone marrow failure and leukemia predisposition due to transcriptional and DNA repair defects
.
Cell Reports
.
2014
;
8
(
3
):
767
-
782
.
83.
Sun
W
,
Downing
JR
.
Haploinsufficiency of AML1 results in a decrease in the number of LTR-HSCs while simultaneously inducing an increase in more mature progenitors
.
Blood
.
2004
;
104
(
12
):
3565
-
3572
.
84.
Ng
KP
,
Hu
Z
,
Ebrahem
Q
,
Negrotto
S
,
Lausen
J
,
Saunthararajah
Y
.
Runx1 deficiency permits granulocyte lineage commitment but impairs subsequent maturation
.
Oncogenesis
.
2013
;
2
:
e78
.
85.
Chin
DW
,
Sakurai
M
,
Nah
GS
, et al
.
RUNX1 haploinsufficiency results in granulocyte colony-stimulating factor hypersensitivity
.
Blood Cancer J
.
2016
;
6
:
e379
.
86.
Song
WJ
,
Sullivan
MG
,
Legare
RD
, et al
.
Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia
.
Nat Genet
.
1999
;
23
(
2
):
166
-
175
.
87.
Cai
X
,
Gao
L
,
Teng
L
, et al
.
Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells
.
Cell Stem Cell
.
2015
;
17
(
2
):
165
-
177
.
88.
Egawa
T
,
Tillman
RE
,
Naoe
Y
,
Taniuchi
I
,
Littman
DR
.
The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells
.
J Exp Med
.
2007
;
204
(
8
):
1945
-
1957
.
89.
Setoguchi
R
,
Tachibana
M
,
Naoe
Y
, et al
.
Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development
.
Science
.
2008
;
319
(
5864
):
822
-
825
.
90.
Woolf
E
,
Xiao
C
,
Fainaru
O
, et al
.
Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis
.
Proc Natl Acad Sci USA
.
2003
;
100
(
13
):
7731
-
7736
.
91.
Taniuchi
I
,
Osato
M
,
Egawa
T
, et al
.
Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development
.
Cell
.
2002
;
111
(
5
):
621
-
633
.
92.
Satpathy
AT
,
Briseño
CG
,
Cai
X
, et al
.
Runx1 and Cbfβ regulate the development of Flt3+ dendritic cell progenitors and restrict myeloproliferative disorder
.
Blood
.
2014
;
123
(
19
):
2968
-
2977
.
93.
Ebihara
T
,
Song
C
,
Ryu
SH
, et al
.
Runx3 specifies lineage commitment of innate lymphoid cells
.
Nat Immunol
.
2015
;
16
(
11
):
1124
-
1133
.
94.
Kuo
YH
,
Zaidi
SK
,
Gornostaeva
S
,
Komori
T
,
Stein
GS
,
Castilla
LH
.
Runx2 induces acute myeloid leukemia in cooperation with Cbfbeta-SMMHC in mice
.
Blood
.
2009
;
113
(
14
):
3323
-
3332
.
95.
Lausen
J
.
Contributions of the histone arginine methyltransferase PRMT6 to the epigenetic function of RUNX1
.
Crit Rev Eukaryot Gene Expr
.
2013
;
23
(
3
):
265
-
274
.
96.
Lichtinger
M
,
Hoogenkamp
M
,
Krysinska
H
,
Ingram
R
,
Bonifer
C
.
Chromatin regulation by RUNX1
.
Blood Cells Mol Dis
.
2010
;
44
(
4
):
287
-
290
.
97.
Obier
N
,
Bonifer
C
.
Chromatin programming by developmentally regulated transcription factors: lessons from the study of haematopoietic stem cell specification and differentiation
.
FEBS Lett
.
2016
;
590
(
22
):
4105
-
4115
.
98.
Swiers
G
,
de Bruijn
M
,
Speck
NA
.
Hematopoietic stem cell emergence in the conceptus and the role of Runx1
.
Int J Dev Biol
.
2010
;
54
(
6-7
):
1151
-
1163
.
99.
Hoogenkamp
M
,
Lichtinger
M
,
Krysinska
H
, et al
.
Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program
.
Blood
.
2009
;
114
(
2
):
299
-
309
.
100.
Huang
G
,
Zhang
P
,
Hirai
H
, et al
.
PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis
.
Nat Genet
.
2008
;
40
(
1
):
51
-
60
.
101.
Lichtinger
M
,
Ingram
R
,
Hannah
R
, et al
.
RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis
.
EMBO J
.
2012
;
31
(
22
):
4318
-
4333
.
102.
Lie-A-Ling
M
,
Marinopoulou
E
,
Li
Y
, et al
.
RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence
.
Blood
.
2014
;
124
(
11
):
e11
-
e20
.
103.
Lancrin
C
,
Mazan
M
,
Stefanska
M
, et al
.
GFI1 and GFI1B control the loss of endothelial identity of hemogenic endothelium during hematopoietic commitment
.
Blood
.
2012
;
120
(
2
):
314
-
322
.
104.
Thambyrajah
R
,
Mazan
M
,
Patel
R
, et al
.
GFI1 proteins orchestrate the emergence of haematopoietic stem cells through recruitment of LSD1
.
Nat Cell Biol
.
2016
;
18
(
1
):
21
-
32
.
105.
Neel
BG
,
Speck
NA
.
Tyrosyl phosphorylation toggles a Runx1 switch
.
Genes Dev
.
2012
;
26
(
14
):
1520
-
1526
.
106.
Ghozi
MC
,
Bernstein
Y
,
Negreanu
V
,
Levanon
D
,
Groner
Y
.
Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions
.
Proc Natl Acad Sci USA
.
1996
;
93
(
5
):
1935
-
1940
.
107.
Telfer
JC
,
Rothenberg
EV
.
Expression and function of a stem cell promoter for the murine CBFalpha2 gene: distinct roles and regulation in natural killer and T cell development
.
Dev Biol
.
2001
;
229
(
2
):
363
-
382
.
108.
Bae
SC
,
Ogawa
E
,
Maruyama
M
, et al
.
PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials
.
Mol Cell Biol
.
1994
;
14
(
5
):
3242
-
3252
.
109.
Bae
SC
,
Yamaguchi-Iwai
Y
,
Ogawa
E
, et al
.
Isolation of PEBP2 alpha B cDNA representing the mouse homolog of human acute myeloid leukemia gene, AML1
.
Oncogene
.
1993
;
8
(
3
):
809
-
814
.
110.
Fukushima-Nakase
Y
,
Naoe
Y
,
Taniuchi
I
,
Hosoi
H
,
Sugimoto
T
,
Okuda
T
.
Shared and distinct roles mediated through C-terminal subdomains of acute myeloid leukemia/Runt-related transcription factor molecules in murine development
.
Blood
.
2005
;
105
(
11
):
4298
-
4307
.
111.
Bangsow
C
,
Rubins
N
,
Glusman
G
, et al
.
The RUNX3 gene—sequence, structure and regulated expression
.
Gene
.
2001
;
279
(
2
):
221
-
232
.
112.
Bone
KR
,
Gruper
Y
,
Goldenberg
D
,
Levanon
D
,
Groner
Y
.
Translation regulation of Runx3
.
Blood Cells Mol Dis
.
2010
;
45
(
2
):
112
-
116
.
113.
Terry
A
,
Kilbey
A
,
Vaillant
F
, et al
.
Conservation and expression of an alternative 3′ exon of Runx2 encoding a novel proline-rich C-terminal domain
.
Gene
.
2004
;
336
(
1
):
115
-
125
.
114.
Fujita
Y
,
Nishimura
M
,
Taniwaki
M
,
Abe
T
,
Okuda
T
.
Identification of an alternatively spliced form of the mouse AML1/RUNX1 gene transcript AML1c and its expression in early hematopoietic development
.
Biochem Biophys Res Commun
.
2001
;
281
(
5
):
1248
-
1255
.
115.
Levanon
D
,
Bernstein
Y
,
Negreanu
V
, et al
.
A large variety of alternatively spliced and differentially expressed mRNAs are encoded by the human acute myeloid leukemia gene AML1
.
DNA Cell Biol
.
1996
;
15
(
3
):
175
-
185
.
116.
Pozner
A
,
Goldenberg
D
,
Negreanu
V
, et al
.
Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms
.
Mol Cell Biol
.
2000
;
20
(
7
):
2297
-
2307
.
117.
Ben-Ami
O
,
Pencovich
N
,
Lotem
J
,
Levanon
D
,
Groner
Y
.
A regulatory interplay between miR-27a and Runx1 during megakaryopoiesis
.
Proc Natl Acad Sci USA
.
2009
;
106
(
1
):
238
-
243
.
118.
Rossetti
S
,
Sacchi
N
.
RUNX1: A microRNA hub in normal and malignant hematopoiesis
.
Int J Mol Sci
.
2013
;
14
(
1
):
1566
-
1588
.
119.
Levanon
D
,
Glusman
G
,
Bangsow
T
, et al
.
Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1
.
Gene
.
2001
;
262
(
1-2
):
23
-
33
.
120.
Miyoshi
H
,
Shimizu
K
,
Kozu
T
,
Maseki
N
,
Kaneko
Y
,
Ohki
M
.
t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1
.
Proc Natl Acad Sci USA
.
1991
;
88
(
23
):
10431
-
10434
.
121.
Goyama
S
,
Yamaguchi
Y
,
Imai
Y
, et al
.
The transcriptionally active form of AML1 is required for hematopoietic rescue of the AML1-deficient embryonic para-aortic splanchnopleural (P-Sp) region
.
Blood
.
2004
;
104
(
12
):
3558
-
3564
.
122.
Okuda
T
,
Takeda
K
,
Fujita
Y
, et al
.
Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy
.
Mol Cell Biol
.
2000
;
20
(
1
):
319
-
328
.
123.
Tanaka
T
,
Tanaka
K
,
Ogawa
S
, et al
.
An acute myeloid leukemia gene, AML1, regulates hemopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms
.
EMBO J
.
1995
;
14
(
2
):
341
-
350
.
124.
Tanaka
T
,
Tanaka
K
,
Ogawa
S
, et al
.
An acute myeloid leukemia gene, AML1, regulates transcriptional activation and hemopoietic myeloid cell differentiation antagonistically by two alternative spliced forms
.
Leukemia
.
1997
;
11
(
Suppl 3
):
299
-
302
.
125.
Tsuzuki
S
,
Seto
M
.
Expansion of functionally defined mouse hematopoietic stem and progenitor cells by a short isoform of RUNX1/AML1
.
Blood
.
2012
;
119
(
3
):
727
-
735
.
126.
Tsuzuki
S
,
Hong
D
,
Gupta
R
,
Matsuo
K
,
Seto
M
,
Enver
T
.
Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1
.
PLoS Med
.
2007
;
4
(
5
):
e172
.
127.
Komeno
Y
,
Yan
M
,
Matsuura
S
, et al
.
Runx1 exon 6-related alternative splicing isoforms differentially regulate hematopoiesis in mice
.
Blood
.
2014
;
123
(
24
):
3760
-
3769
.
128.
Tsuji
K
,
Noda
M
.
Identification and expression of a novel 3′-exon of mouse Runx1/Pebp2alphaB/Cbfa2/AML1 gene
.
Biochem Biophys Res Commun
.
2000
;
274
(
1
):
171
-
176
.
129.
Challen
GA
,
Goodell
MA
.
Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells
.
Exp Hematol
.
2010
;
38
(
5
):
403
-
416
.
130.
Brady
G
,
Elgueta Karstegl
C
,
Farrell
PJ
.
Novel function of the unique N-terminal region of RUNX1c in B cell growth regulation
.
Nucleic Acids Res
.
2013
;
41
(
3
):
1555
-
1568
.
131.
Bee
T
,
Ashley
EL
,
Bickley
SR
, et al
.
The mouse Runx1 +23 hematopoietic stem cell enhancer confers hematopoietic specificity to both Runx1 promoters
.
Blood
.
2009
;
113
(
21
):
5121
-
5124
.
132.
Pimanda
JE
,
Donaldson
IJ
,
de Bruijn
MF
, et al
.
The SCL transcriptional network and BMP signaling pathway interact to regulate RUNX1 activity
.
Proc Natl Acad Sci USA
.
2007
;
104
(
3
):
840
-
845
.
133.
Ugarte
GD
,
Vargas
MF
,
Medina
MA
, et al
.
Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells
.
Blood
.
2015
;
126
(
15
):
1785
-
1789
.
134.
Sroczynska
P
,
Lancrin
C
,
Kouskoff
V
,
Lacaud
G
.
The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis
.
Blood
.
2009
;
114
(
26
):
5279
-
5289
.
135.
Bee
T
,
Swiers
G
,
Muroi
S
, et al
.
Nonredundant roles for Runx1 alternative promoters reflect their activity at discrete stages of developmental hematopoiesis
.
Blood
.
2010
;
115
(
15
):
3042
-
3050
.
136.
Pozner
A
,
Lotem
J
,
Xiao
C
, et al
.
Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis
.
BMC Dev Biol
.
2007
;
7
:
84
.
137.
Ditadi
A
,
Sturgeon
CM
,
Tober
J
, et al
.
Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages
.
Nat Cell Biol
.
2015
;
17
(
5
):
580
-
591
.
138.
Ng
ES
,
Azzola
L
,
Bruveris
FF
, et al
.
Differentiation of human embryonic stem cells to HOXA(+) hemogenic vasculature that resembles the aorta-gonad-mesonephros
.
Nat Biotechnol
.
2016
;
34
(
11
):
1168
-
1179
.
139.
Draper
JE
,
Sroczynska
P
,
Tsoulaki
O
, et al
.
RUNX1B expression is highly heterogeneous and distinguishes megakaryocytic and erythroid lineage fate in adult mouse hematopoiesis
.
PLoS Genet
.
2016
;
12
(
1
):
e1005814
.
140.
Mukai
K
,
BenBarak
MJ
,
Tachibana
M
, et al
.
Critical role of P1-Runx1 in mouse basophil development
.
Blood
.
2012
;
120
(
1
):
76
-
85
.
141.
Marsman
J
,
O’Neill
AC
,
Kao
BR
, et al
.
Cohesin and CTCF differentially regulate spatiotemporal runx1 expression during zebrafish development
.
Biochim Biophys Acta
.
2014
;
1839
(
1
):
50
-
61
.
142.
Nottingham
WT
,
Jarratt
A
,
Burgess
M
, et al
.
Runx1-mediated hematopoietic stem-cell emergence is controlled by a Gata/Ets/SCL-regulated enhancer
.
Blood
.
2007
;
110
(
13
):
4188
-
4197
.
143.
Ng
CE
,
Yokomizo
T
,
Yamashita
N
, et al
.
A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells
.
Stem Cells
.
2010
;
28
(
10
):
1869
-
1881
.
144.
Tamplin
OJ
,
Durand
EM
,
Carr
LA
, et al
.
Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche
.
Cell
.
2015
;
160
(
1-2
):
241
-
252
.
145.
Zhang
P
,
He
Q
,
Chen
D
, et al
.
G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition
.
Cell Res
.
2015
;
25
(
10
):
1093
-
1107
.
146.
Ferrell
PI
,
Xi
J
,
Ma
C
,
Adlakha
M
,
Kaufman
DS
.
The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells
.
Stem Cells
.
2015
;
33
(
4
):
1130
-
1141
.
147.
Schütte
J
,
Wang
H
,
Antoniou
S
, et al
.
An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability
.
eLife
.
2016
;
5
:
e11469
.